Streptococcus mutans is the primary etiological agent of dental caries (cavities), but the bacterium also causes endocarditis and is strongly associated with cardiovascular disease and stroke. The vast majority of the factors that allow S. mutans to cause disease are located in the cytoplasmic membrane or are secreted outside of the cell. Proper biogenesis of the cell envelope of S. mutans requires two paralogous YidC proteins of the universally conserved YidC/Oxa/Alb3 family. Deletion of either yidC gene results in diminished virulence of S. mutans in a rat caries model, while deletion of both genes is lethal. Moreover, stress tolerance, H+-ATPase activity, and biofilm formation are decreased in yidC mutants, with more severe effects observed upon deletion of yidC2 compared to yidC1. Additionally, there is strong evidence that yidC1 and yidC2 are differentially regulated in lactic acid bacteria. In a recent collaborative effort, we sequenced and analyzed the genomes of 57 clinical isolates of S. mutans, demonstrating that the pan-, and core-genomes contain approximately 3300 and 1400 genes, respectively. This study also predicted a number of small RNAs in S. mutans, and through RNA-deep sequencing we determined that some of the sRNAs are differentially expressed. As part of the postdoctoral training of the applicant, an extensive phenotypic characterization was performed on 15 of the sequenced strains, and it was demonstrated that there is substantial diversity in gene content and virulence potential within the species S. mutans. The major scientific goals of this proposal are to dissect the molecular basis for regulation of yidC1 and yidC2, and to provide a comprehensive understanding of the roles of these proteins in stress adaptation and cellular physiology across the species S. mutans. To accomplish these goals, the following Specific Aims are delineated: Aim 1. Determine the mechanisms of regulation of yidC1 and yidC2 in response to growth domain, and to environmental influences that are known to induce membrane stress and to affect persistence and pathogenesis by S. mutans. Aim 2. Investigate the role of small RNAs, a ribonuclease, and an RNA binding protein encoded within the yidC gene clusters in the regulation of yidC expression and function of the YidC proteins Aim 3. Determine how deletion of yidC1 or yidC2 affects the transcriptomes of phenotypically and genetically-diverse isolates of S. mutans using RNA-deep sequencing. The completion of this project will also allow the applicant to receive critical training in the areas of gene regulation and genome-scale analytical techniques, while obtaining the mentoring and career development counseling to facilitate her transition to an independent research career. The data generated will provide essential new information that can be used to design novel and more effective therapies to combat dental caries and other bacterial infections.